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Abstract

Curcumin has been shown to exert potential antitumor activity in vitro and in vivo involved in multiple signaling pathways. However, the application of curcumin is still limited because of its poor hydrophilicity and low bio-availability. In the present study, we investigated the therapeutic effects of a novel and water soluble bis(hydroxymethyl) alkanoate curcuminoid derivative, MTH-3, on human breast adenocarcinoma MDA-MB-231 cells. This study investigated the effect of MTH-3 on cell viability, cell cycle and induction of autophagy and apoptosis in MDA-MB-231 cells. After 24-h treatment with MTH-3, a concentration-dependent decrease in MDA-MB-231 cell viability was observed, and the IC50 value was 5.37±1.22 µM. MTH-3 significantly triggered G2/M phase arrest and apoptosis in MDA-MB-231 cells. Within a 24-h treatment, MTH-3 decreased the CDK1 activity by decreasing CDK1 and cyclin B1 protein levels. MTH-3-induced apoptosis was further confirmed by morphological assessment and annexin V/PI staining assay. Induction of apoptosis caused by MTH-3 was accompanied by an apparent increase of DR3, DR5 and FADD and, as well as a marked decrease of Bcl-2 and Bcl-xL protein expression. MTH-3 also decreased the protein levels of Ero1, PDI, PERK and calnexin, as well as increased the expression of IRE1α, CHOP and Bip that consequently led to ER stress and MDA-MB-231 cell apoptosis. In addition, MTH-3-treated cells were involved in the autophagic process and cleavage of LC3B was observed. MTH-3 enhanced the protein levels of LC3B, Atg5, Atg7, Atg12, p62 and Beclin-1 in MDA-MB-231 cells. Finally, DNA microarray was carried out to investigate the level changes of gene expression modulated by MTH-3 in MDA-MB-231 cells. Taken together, our results suggest that MTH-3 might be a novel therapeutic agent for the treatment of triple-negative breast cancer in the near future.

Introduction

Breast cancer is the second leading cause of death
in women and has approximately 1 million new cases per year
worldwide (1,2). Breast cancer patients develop
metastasis eventually leading to poor prognosis (3). Triple-negative breast cancer (TNBC)
accounts for 12–20% of all breast cancer (4). It has more aggressive disease
progress and worse prognosis (5).
TNBC characteristics are the lack of expression of estrogen
receptor (ER), progesterone receptor (PR) and the lack of
overexpression of HER-2 (4,6).
TNBC is resistance to anti-hormone therapies and HER-2-aiming
target therapies (7,8). Treatment of TNBC remains a great
clinical challenge because of the lack of targeting agents and
limited therapeutic options (8,9).

Curcumin has been used in traditional Chinese
medicine for a long time in Taiwan, China and India (10). The pharmacological effects of
curcumin include anti-amyloid (11), anti-bacterial (12), anti-depressant (13), anti-inflammatory (14), anti-oxidant (15), anti-diabetes (16) and anticancer properties (17,18).
In addition, curcumin has been found to affect several anticancer
signaling pathways such as inhibition of cancer cell proliferation
(19,20) and induction of cell cycle arrest
(21), apoptosis (22) or autophagy (23). Specifically, the phase II and III
clinical trial of curcumin was advocated for use in patients with
colon and pancreatic cancers (24,25),
but its low water solubility exerts poor bioavailability and
primary limiting factors (low efficacy and safety) (26,27).
To improve these issues, we designed and developed a novel
bis(hydroxymethyl) alkanoate curcuminoid derivative, MTH-3
(Fig. 1). In our previous studies,
novel bis(hydroxymethyl) alkanoate curcuminoid derivatives were
shown to exhibit antitumor effects on triple-negative breast cancer
cells and in a xenograft animal experiment (28). The aim of the present study was to
characterize the property of MTH-3 and to clarify the molecular
mechanism of MTH-3 in human breast adenocarcinoma MDA-MB-231 cells
in vitro.

Cell culture

The human breast adenocarcinoma cell line MDA-MB-231
was purchased from the Bioresource Collection and Research Center
(BCRC; Hsinchu, Taiwan). Cells were cultured in Leibovitz's L-15
medium with 10% FBS and 1% penicillin-streptomycin (100 Units/ml
penicillin and 100 μg/ml streptomycin) in an incubator under
95% air and 5% CO2 at 37°C.

Cell viability assay and morphologic
changes

Cell viability was evaluated by the reduction in MTT
to yield blue formazan. MDA-MB-231 cells (1×104
cells/well) in 96-well plates were allowed to attach overnight and
then treated with different concentrations (1, 3, 5 and 10
μM) of MTH-3 for 24 h. After treatments, MTT solution was
added to each well (a final concentration of 0.5 μg/ml), and
then the plates were incubated for another 4 h. The medium was
removed, blue formazan was dissolved in dimethyl sulfoxide (DMSO),
and the absorbance was read at 570 nm as previously described
(29). For trypan blue exclusion
assay, cells were collected after 1, 3, 5 and 10 μM of MTH-3
exposure, stained with 0.4% trypan blue and then counted on a
hemocytometer under a microscope. For morphological observation,
cells were visualized and photographed using a phase-contrast
microscope equipped with a digital camera (Leica Microsystems GmbH,
Wetzlar, Germany) as in previous reports (26,30).

Distribution of cell cycle analysis

MDA-MB-231 cells (2×105 cells/well) in
12-well plates were exposed to 10 μM MTH-3. After a 24-h
treatment, cells were harvested and fixed gently by putting 70%
ethanol at 4°C overnight before being stained with PI solution (40
μg/ml PI and 0.1 mg/ml RNase and 0.1% Triton X-100) in the
dark for 30 min as previously described (31). The cells were analyzed for the cell
cycle distribution with a flow cytometer (FACSCalibur; BD
Biosciences, San Jose, CA, USA).

CDK1 kinase assay

CDK1 kinase activity was analyzed according to the
manufacturer's protocol (CycLex Cdc2-Cyclin B Kinase Assay kit; MBL
International Corp., Woburn, MA, USA). The ability of CDK1 kinase
from MDA-MB-231 cell extracts prepared from each treatment of 10
μM MTH-3 for 4, 8, 16 and 24 h was measured as previously
described (32,33).

Immunofluorescence staining

MDA-MB-231 cells (2×106 cells/dish) were
grown on sterile coverslips placed in a 10-cm dish. After 10
μM MTH-3 treatment, cells were fixed with 4%
paraformaldehyde and permeabilized with 0.2% Triton X-100 in
phosphate-buffered saline (PBS). After blocking with 2% bovine
serum albumin (BSA) in PBS, LC3B and p62 were detected using
anti-LC3B and anti-p62 antibody followed by reaction with FITC- or
PE-conjugated secondary antibody (BD Biosciences). Coverslips were
mounted on glass slides with ProLong Gold Antifade reagents (Thermo
Fisher Scientific) containing DAPI, and fluorescent image was taken
on a Leica Microsystems TCS SP2 Confocal Spectral microscope as
detailed by Lu et al (39).

cDNA microarray analysis

MDA-MB-231 cells were incubated with or without 10
μM MTH-3 for 24 h. After exposure, cell pellets were
collected, and the total RNA from each treatment was purified using
the Qiagen RNeasy Mini kit (Qiagen, Valencia, CA, USA). RNA purity
was determined to check the quality at 260/280 nm using a NanoDrop
1000 spectrophotometer (Thermo Fisher Scientific). mRNA was
amplified and labeled using the GeneChip WT Sense Target Labeling
and Control Reagents kit (Affymetrix, Santa Clara, CA, USA) for
expression analysis. The synthesized cDNA was labeled with
fluorescence and then hybridized for 17 h using GeneChip Human Gene
1.0 ST array (Affymetrix) to determine microarray hybridization
following the manufacturer's protocols. The arrays were
subsequently washed using GeneChip Fluidics Station 450
(Affymetrix), stained with streptavidin-phycoerythrin (GeneChip
Hybridization, Wash and Stain kit; Affymetrix) and scanned on a
GeneChip Scanner 3000 (Affymetrix). The localized concentrations of
fluorescent molecules were quantitated and analyzed using
Expression Console Software (Affymetrix) with default RMA
parameters as previously described (40). The gene expression level of a
2.5-fold change (log2 ratio) was considered a difference in
MTH-3-treated cells in vitro (41,42).

Statistical analysis

Data are presented as the mean ± SD for three
separate experiment. Differences among the groups were considered
to be significant at P<0.05 using ANOVA followed by the Duncan's
test.

Results

At first, the effect of MTH-3 on the viability of
MDA-MB-231 cells was investigated using the MTT and trypan blue
exclusion assays. MTH-3 at 1, 3, 5 and 10 μM significantly
reduced the viability of MDA-MB-231 cells by 98.94±2.26,
89.57±2.07, 69.57±4.13 and 59.6±4.04%, respectively (Fig. 2A). Importantly, the cell viability
reduction after 30 μM MTH-3 challenge is 34.23±3.31%. This
effect is in a concentration-dependent manner. Data from
morphological observation revealed that MTH-3 treatment at 10
μM caused obvious MDA-MB-231 cell apoptosis and autophagy
with characteristics, including cytoplasmic membrane blebbing, cell
shrinkage and autophagic vacuoles (Fig. 2B). Based on these findings and
gaining effective evidence of cell death, we selected MTH-3 at 10
μM for the majority of the experiments in MDA-MB-231
cells.

The effects of MTH-3 on apoptosis-related proteins
in MDA-MB-231 cells were investigated. Our results demonstrated
that MTH-3 upregulated the levels of DR5 and FADD, and it
downregulated the levels of Bcl-2 and Bcl-xL (Fig. 5A). Furthermore, our findings also
revealed that MTH-3 markedly increased the levels of CHOP and Bip,
as well as decreased the levels of Ero1, PDI, PERK, calnexin and
IRE1α (Fig. 5B). These results
suggest that MTH-3 induced apoptosis through death receptor
(extrinsic pathway) and mitochondria (intrinsic pathway)-dependent
pathways and possibly by modulating ER stress mechanism in
MDA-MB-231 cells.

MTH-3 alters the levels of
autophagy-associated proteins in MDA-MB-231 cells

Based on the results of autophagy, its related
signals were further employed by immunoblotting analysis. MTH-3
treatment induced the levels of Atg5, Atg7, Atg12, Beclin-1, p62
and LC3B in a time-dependent manner (Fig. 7). These data demonstrated that
MTH-3 induced autophagy by activating Atg family proteins in
MDA-MB-231 cells.

After treatment with 10 μM MTH-3 for 24 h,
cells were collected, and cDNA microarray analysis was performed.
The analysis showed that 97 genes (69 genes, upregulated; 28 genes,
down-regulated) were expressed at least by 2.5-fold compared with
the untreated control (Table I).
The top alteration in gene expression scored by the number of
pathway networks from GeneGo analysis program (Fig. 8). These genes may also be involved
in cell death and cytotoxic responses in MTH-3-treated MDA-MB-231
cells.

Discussion

Previous studies have demonstrated the anticancer
potential of curcumin in regulating cell cycle, autophagy,
apoptosis and survival, proliferation, angiogenesis, invasion and
metastasis (19–23). Guan et al (43) demonstrated that curcumin reduced
Akt kinase in MDA-MB-231 cells accompanied by a decrease in cell
proliferation and migration as well as an increase in autophagic
activity; moreover, AMPK-mediated activation of autophagy
contributes to anticancer effects through Akt degradation. In the
present study, we also checked the growth inhibition effect of
curcumin on MDA-MB-231 cells. Our data indicated that the half
maximal inhibitory concentration (IC50) value of
curcumin on MDA-MB-231 cells is 38.77±3.35 μM. Strikingly,
the IC50 value of MTH-3 on MDA-MB-231 cells is 5.37±1.22
μM (data not shown). Our results demonstrated that the MTH-3
had highly cytotoxic effects on MDA-MB-231 cells. Moreover, we also
found that MTH-3 was non-cytotoxic on non-tumorigenic epithelial
mammary MCF10A cells and human skin fibroblast Detroit 551 cells,
respectively (data not shown). These are only preliminary data and
further study is needed to validate the findings.

There are no reports regarding that the effects of
MTH-3 on cell cycle arrest, autophagy and apoptosis and associated
gene expression in human breast cancer cells. This study is first
to demonstrate that MTH-3 induced cytotoxic effect on induction of
G2/M phase arrest, autophagy and apoptosis in human
breast adenocarcinoma MDA-MB-231 cells. The data demonstrated that
MTH-3 induced growth inhibitory effects through G2/M
phase arrest, apoptosis and autophagy in MDA-MB-231 cells. Our
results showed that MTH-3 induced G2/M phase arrest
through regulating cyclin B1 and CDK1 signaling. G2/M
phase progression has been reported to regulate CDK1 and CDK2
kinases that are activated primarily in association with cyclins A
and B (44). Furthermore, MTH-3
inhibited the CDK1 activity and the protein expression of CDK1 in
MDA-MB-231 cells. However, neither effect is positively correlated
because CDK1 activity might be involved in kinase activation rather
than CDK1/cdc2 protein level (32,33).
Previous studies also demonstrated that curcumin inhibited cell
proliferation through induction of G0/G1 phase arrest of
cancer cells (45,46), but our finding indicated that MTH-3
induced G2/M phase arrest upon different types of cancer
cell lines. However, the results are in agreement with previous
studies to show that curcumin inhibited cell proliferation by
inducing G2/M phase arrest in human glioblastoma U87
cells (47) and in Bcl-2
overex-pressed MCF-7 cells (48).
Further research is required to verify the mechanism of MTH-3
action in different breast cancer cell lines (such as MCF-7 and
MDA-MB-453 cells).

It is well documented that apoptosis plays an
important role in the maintenance of tissue homeostasis for the
elimination of excessive cells (49,50).
Induction of apoptosis of cancer cells by anticancer drugs such as
etoposide, cisplatin and paclitaxel have been used for treatment of
cancer in target cells (51).
Apoptosis-associated signaling pathways include extrinsic (death
receptor), intrinsic (mitochondria-dependent) and ER stress
(unfolded protein response) signals (52,53).
Our results demonstrated that MTH-3 promoted the protein levels of
DR5, and FADD and downregulated the levels of Bcl-2 and Bcl-xL in
MDA-MB-231 cells. MTH-3 also promoted the protein levels of CHOP
and Bip, and it reduced the levels of Ero1, PDI, PERK, calnexin and
IRE1α in MDA-MB-231 cells. Our novel findings suggest that both
extrinsic and intrinsic pathways, and ER stress signals were
involved in MTH-3-treated cells in vitro. This agrees with a
previous study reporting that the major targets of apoptotic
initiation are mediated by dysfunction of cellular organelles
(mitochondria, ER, lysosomes and golgi apparatus) (54).

In conclusion, the molecular signaling pathways are
summarized in Fig. 9. This study
is the first report to provide an approach regarding the
bis(hydroxymethyl) alkanoate curcuminoid derivative, MTH-3 tends to
inhibit human breast adenocarcinoma MDA-MB-231 cells. Based on the
presented novel findings, the efficacy of MTH-3 might be sufficient
to further investigate the potential of breast cancer
treatment.

Acknowledgments

The present study was supported by research grants
from the National Science Council of the Republic of China awarded
to S.-C.K. and by China Medical University under the Aim for Top
University Plan of the Ministry of Education, Taiwan
(CHM106-2).